√ Why We Shouldn’T View The Human Embryo As A Gizmo Even In The Crispr Era

My first job in science was as a lab technician at UCSD School of Medicine and a big part of that job was growing cells called HUVECs or human umbilical vein endothelial cells. We isolated and grew the HUVECs from umbilical cords that we retrieved from the maternity ward of the UCSD hospital, which first entailed getting the placenta and attached umbilical cord as our starting material.

Some people viewed this material as “gross”, but as a newbie scientist I was kind of in awe of the whole process and it made me think about human development starting with human embryos and leading up to birth of babies in a new way. Those HUVECs were integral to proper human development and it was cool even if challenging to grow them.

Now about 25 years later, after working with dozens of human cell types, I have been thinking more about human embryos the last few years, especially as relates to CRISPR, even though I don’t do research on them in my lab.

How should we view a human embryo, both overall societally and within a scientific context?

Is a human embryo just the same as any other research material?

If a lab is working with a 100-cell human embryo is there some kind of equivalency between that and say the dish next to it in that same lab with a cluster of 100 human stem cells? What about 100 human skin cells grown in a lab?

Is a human cell just a human cell?

I don’t think so on either account. However, at times it seems like some research on human embryos (and commentary on it by scientists, philosophers, or others voicing their opinions) reflects a certain nonchalance about it or a sense that no special care or thought was or should be given to this kind of work. I call this a “gizmo” mentality about human embryos. That these are just some “thing” that can be fiddled with and you can always get another one. I believe that kind of mindset should be avoided.

For context, I’m not a particularly religious person so that is not driving my viewpoint here. Also, I personally do not believe that human embryos are people and I’m not a fan of the personhood movement. At the other extreme, human embryos also are not just a collection of random cells either. They aren’t a gizmo.

In my view, research on human embryos, such as introduction of CRISPR into them for gene editing or some other kind of larger scale genetic modification like a “knockout”, requires extra consideration. There should be some advance contemplation about it and ideally consultation with a bioethicist. These extra steps and planning via researchers asking themselves questions and having discussions with others should start early and before you begin the actual embryo research. Better to ask questions early than too late.

For example, where will the human embryos come from and where did the gametes come from to make them?

Were gamete donors or embryo donors properly consented?

Are the embryos “leftovers” from IVF or are they being made purposefully just for research? The latter requires more consideration.

Why are you doing the human embryo research and is there a rock-solid justification for this work? Could you not answer the same research question using mouse embryos for example? You couldn’t address the same research challenge with just stem cells in a dish in the lab?

These aren’t easy questions, but they are important.

Since human gametes and embryos made from them require human donors (and there are risks to donors) and have the potential if implanted to make a human being, you should be extra careful in experimental design, biostats, etc. as well. Also, you need to think in advance about what you will do with the experimental embryos once the project is done if some are to be frozen.

This is all a lot more complicated than just experimenting on every day human cells in a dish, but the extra steps are needed because again human embryos are not your average group of 100 cells. Going the extra mile to do human embryo research properly lowers risks to others and to the research field.

Further, consider how your work might be portrayed in the media, especially if you have some future clinical intent with your human embryo project in years to come such as trying to prevent a genetic disease. Being proactive and thoughtful can help you avoid the risk of being at the center of some controversy. If you have some long-range clinical intent for your work, you should also be very cautious in publicly discussing that and always discuss existing alternatives like PGD for prevention of genetic diseases in future offspring.

It’s encouraging to see that some labs publishing high-profile human embryo papers recently have apparently given it quite a bit of thought and there has been advance institutional review, but some other reports out there and/or statements in the media from those doing such research or individuals commenting on it are concerning. There are clearly some important gaps that need to be addressed in how this work is approached and discussed. Some gizmo mentality can creep in at times in discussing actual human embryo research.

We should also be conscious that as human embryo work such as that using CRISPR becomes more commonplace, as it seems to be trending lately, that doesn’t change the core questions and bioethical concerns. Just because someone for example has CRISPR’d 500 human embryos doesn’t mean those embryos should be viewed as commonplace research materials. Extraordinary mindfulness is always going to be needed for this kind of research even if it becomes more commonly conducted.

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√ Mitalipov Rebuts Crispr Pub Critiques: Strengthens Case, But Puzzles Remain

Remember that Shoukhrat Mitalipov lab paper on the use of CRISPR in human embryos? It’s back in the news.

Ma, et al. 2018 Nature, FIgure 1

One of the biggest stories of 2017 centered on a Nature paper (Ma, et al., see my quick, initial review shortly after it was published here) from Mitalipov’s lab claiming both efficient repair of a disease-causing mutation in human embryos via CRISPR-Cas9 gene editing and that the correction happened at the MYBPC3 gene via an unexpected mechanism using the normal maternal chromosome as a template (the mutation was paternal) via so-called “interhomolog repair.”

Then another group’s preprint (Egli, et al.) upped the intensity by challenging the Ma paper’s central conclusions and proposing alternative explanations that some of us (see my posts here and here) found quite plausible.

Now Mitalipov’s team has published a rebuttal of a sort to the critiques in Nature along with the finalized paper from Egli, et al. and a comment from another group led by Paul Thomas.

Now everything’s crystal clear, right?

Not really, but a quick take-home is that Mitalipov’s group has convinced me that it is more likely than not that some interhomolog repair happened in some embryos at least, but things are still somewhat up in the air.

I won’t rehash all the main issues that led to questioning of the Ma paper, but just try to be concise about it and how Mitalipov’s group responded.

Chopped to be undetectable rather than fixed? First, there was some concern that rather than really repairing the mutant allele via CRISPR-Cas9 and the maternal WT chromosome as template, CRISPR-Cas9 had further damaged the allele rendering it undetectable by PCR/sequencing and hence giving the incorrect appearance of an entirely WT embryo. Mitalipov’s team responded to this mainly with PCR data in Figure 1 of their rebuttal. They used a series of increasingly widely spaced primers to generate an expanding set of amplicons using genomic DNA from embryos/blastomeres or ES cells as templates. If there were alleles with large deletions, they reasoned this approach would detect them. On the 1% agarose gels shown, there are no intense small product bands that one might expect with certain primer sets if deletions were present. However, there are some important caveats here. The data in Figure 1 are frankly not so clear nor precise, and pretty messy in some cases (see above). Also, there are in fact, as the authors acknowledge, extra bands here and there. While they dismiss these extra bands without much of a look, they could have meaning. Surprisingly, they also did not sequence their dominant amplicons in Figure 1, which could have probably made things clearer.

Lost rather than fixed? Second, it was also pointed out by Egli, et al. that it was possible that the mutant paternal allele wasn’t fixed, but rather lost either via chromosome loss of parthenogenic embryo development. Such loss would mimic the appearance of a “fixed”, only WT embryo. Mitalipov’s team aims to rebut this with various SNP data in their reply now suggesting that there isn’t parthenogenesis and that various embryos and blastomeres did in fact arise from both maternal and paternal contributions. I’m still trying to wrap my head entirely around the data here, but so far I’m thinking it’s generally supporting their claim. There could be other explanations, although I’m not sure how likely they are.

A new, unusual mitotic model of human embryo gene editing raises more puzzles. One of the take-homes from the Mitalipov reply piece is a new model that differs somewhat from the original Ma paper in that they are now arguing for much later gene editing occurring probably at the two-cell stage and via mitosis.

While an intriguing model, it has some issues and puzzling aspects to it.

Mitotic interhomolog repair happens such as in ES cells, but is very rare, while the Ma paper suggests it happens at least in embryos quite often.

If it does happen during mitosis, wouldn’t this sometimes lead to LOH and reduced genetic diversity? I’m not an evolutionary biologist, but it seems like mitotic interhomolog repair in early embryos could cause issues and be inconsistent with our current understanding of how things work.

Perhaps one of the biggest issue is that late CRISPR-Cas9 triggered allelic repair really should lead to a high degree of mosaicism, while Ma, et al. reported little mosaicism.

Also, if CRISPR-Cas9 hangs around dormant but stable in embryos until this late point, why would Ma see such differences in outcomes with the timing of injection of CRISPR-Cas9 machinery either at MII or when they did it much later just before the 2-cell stage? In addition, embryos just tolerate a DSB for all that time prior to mitosis and don’t fix it with NHEJ that would then probably interfere with HDR later?

So overall, Mitalipov’s group has made a more convincing case for interhomolog repair with CRISPR in human embryos, but with so many questions, until we all see more data from various groups, we can’t be sure. One practical challenge is that almost no labs can do this kind of research. Still I’d imagine a year or two from now we’ll have learned more that hopefully will clarify things further.

In the bigger picture I also remain concerned about using CRISPR in human embryos for heritable genetic modification, a notion that was one potential motivation for this work originally.

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√ He Jiankui Didn’T Really Gene Edit Those Girls; He Mutated Them

Did Chinese researcher He Jiankui really CRISPR gene edit the CCR5 gene in two embryos producing twin baby girls? In my opinion the answer is “no”, but probably not for the reason you might think at first.

He proclaims gene edits

He claimed he had made twin baby girls with “gene edits”, which I feel is unethical overall and risky to those babies. He’s announcement also may have been intentionally timed to occur right before an international meeting on human gene (or genome) editing last week too. Glossy YouTube videos from He accompanied all of this as well, raising further suspicions of a PR angle for himself. For these and other reasons, I believe we need a temporary 3-year pause on implantation of CRISPR’d human embryos.

Could He have been stopped and when?

CRISPR gene edit vs mutation. Analogy of what He did to trying to edit a book with a thrown scalpel. Photo Paul Knoepfler

Given the timing and He’s actions, the organizers of the human genome editing meeting could have yanked He’s invitation to speak, but they didn’t. On the whole, that was probably a wise decision so we could all learn more about what supposedly happened.

Others argued He shouldn’t have been allowed to retain that meeting platform since what he did appeared to most likely be unethical on a number of levels. Beyond the questionable science of He’s work, there are still unresolved ethics and transparency questions. As my colleague here at UC Davis Mark Yarborough wrote, in a sense ideally the life sciences community should have hoped to have stopped He much earlier in a different kind of way including via better bioethics pembinaan and a different kind of research culture.

Did He really “gene edit” them?

Some people have wondered if the whole thing might be a fraud in which He didn’t actually make CRISPR’d babies. Based on what we do know, I think most likely He isn’t lying at a core level about having made babies with CRISPR, although we may never know for sure. However, at a common sense level I would still say that He did not gene edit these babies.

What do I mean?

As someone who has been doing genetics for a long time, mostly in mice, I remember that in the pre-CRISPR kurun we called various interventions in genes by other terms: mutations, genetic modifications, gene targeting, etc. Some of us still often use those terms. “Gene editing” sounds more precise and deliberate and benign. Maybe that’s the appeal of this term to some.

In my view as a scientist who uses CRISPR, not all uses of it actually qualify as gene editing and it’s not just semantics. I would argue the term “gene editing” should be reserved only for precise, deliberate changes that justify using versions of the key word “edit”. There’s been far too much loose use of the phrase ‘gene editing” and others like “gene surgery.” This kind of language can be harmful by misinforming people, exaggerating precision, and underestimating risks.

How we write and talk about science is very important (see more on CRISPR metaphors here, work also led by Mark Yarborough). If you think about the “edit” analogy more broadly, would you want your editor to just throw a scalpel or scissors at a specific section of your writing (even if for some weird reason this editor is a very accurate although not perfect thrower) and hope for the best outcome? I’d hardly call that editing.

Gene editing versus mutating

To qualify as a gene edit, He would have had to have been deliberately trying to make one and only one precise change, and have achieved that edit alone. That’s not at all what happened. Instead, it seems He deleted fairly random chunks of a certain region of CCR5 in the human embryos who supposedly became twin girls. None of the He mutations were the same as those occurring naturally in humans who then have substantial resistance to HIV infection.

Even in basic science research using CRISPR, if we make random mutations (what are collectively termed indels, which is short for “Insertions” and “deletions”) should we be calling that “gene editing”? No, we really shouldn’t, even if sometimes those indels are useful and powerful tools. In my lab we have used CRISPR to both make precise gene edits and separately to make indel mutations.

A gene edit is still a type of mutation, but a uniquely precise, deliberate one, while conversely not all mutations are gene edits. Further, not everything done by CRISPR should be called “gene editing.”

Frankly, what He did really was to mutate those twin girls, particularly since he was changing a normal wildtype gene to a mutant form. We should call it what it is so as to be as accurate as possible and think about the reality of the situation.

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